Condensation products of phenol and salicylaldehyde

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames Test, Váliczkó (2016)

Under the conditions of this study, the test material had no mutagenic activity in the applied bacterium tester strains.

Chromosome aberration (2019)

Under the conditions of the study the test material did not induce a significant level of chromosome aberrations in Chinese hamster V79 cells either with or without metabolic activation. Therefore, the test material was considered as not clastogenic in this test system

Mouse Lymphoma Assay (2020)

Under the conditions of the study, treatment with the test material did result in a statistically significant and biologically relevant, dose dependent increase in the mutation frequency in the presence of a rat metabolic activation system (S9 fraction) in Assay 1, the observed effect was repeatable within or between assays. Therefore, reproducible positive effect of the test material was concluded in the performed experiments; overall the test material was considered to be positive with metabolic activation.

Negative results were seen in the experiment in the absence of a rat metabolic activation system (S9 fraction). Statistical differences were not supported by any results above the GEF with the exception of one concentration in Assay 2 without metabolic activation, however in this case excessive cytotoxicity was observed, thus the result would not be considered positive.

The Mouse Lymphoma Assay was considered to be valid and to reflect the real potential of the test material to cause mutations in the cultured mouse cells used in this study.

Overall the test material was considered to be mutagenic.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

02 March 2016 to 24 March 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Version / remarks:

1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

- Histidine requirement in the Salmonella typhimurium strains (Histidine operon).
- Tryptophan requirement in the Escherichia coli strain (Tryptophan operon).

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Details on mammalian cell type (if applicable):

- Source: 21 April 2015, MOLTOX - Molecular Toxicology Inc., Boone, North Carolina, USA.
- Storage: The strains are stored at -80 ± 10 °C in the Culture Collection of the Microbiological Laboratory of the testing facility. Frozen permanent cultures of the tester strains were prepared from fresh, overnight cultures to which DMSO was added as a cryoprotective agent.
- Confirmation of Phenotypes of Tester Strains: The phenotypes of the tester strains used in the bacterial reverse mutation assays with regard to membrane permeability (rfa), UV sensitivity (uvrA and uvrB), ampicillin resistance (amp), as well as spontaneous mutation frequencies are checked regularly according to Ames et al. and Maron and Ames.
- Spontaneous Reversion of Tester Strains: Each test strain reverts spontaneously at a frequency that is characteristic of the strain. Spontaneous reversion of the test strains to histidine (Salmonella typhimurium strains) independence is measured routinely in mutagenicity experiments and expressed as the number of spontaneous revertants per plate.
Historical control values for spontaneous revertants (revertants/plate) for untreated control sample without metabolic activation were in the period of 2011-2014 were as follows: Salmonella typhimurium TA98: 9-46, TA100: 54-210, TA1535: 1-46, TA1537: 1-24.
- Procedure for Growing Cultures: The frozen bacterial cultures were thawed at room temperature and 200 μL inoculum were used to inoculate each 50 mL of Nutrient Broth No.2 for the overnight cultures in the assay. The cultures were incubated for 10-14 hours at 37 °C in a Gyrotory water bath shaker.
- Viability of the Testing Cultures: The viability of each testing culture was determined by plating 0.1 mL of the 10^5, 10^6, 10^7 and 10^8 dilutions prepared by sterile physiological saline on Nutrient Agar plates. The viable cell number of the cultures was determined by manual counting after approximately 24-hour incubation at 37 °C.
- Media: Nutrient Broth No.2 containing: Nutrient Broth No.2 25.0 g and Distilled water q.s. ad 1 000 mL. Sterilisation was performed at 121 °C in an autoclave.

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

- Source: 21 April 2015, MOLTOX - Molecular Toxicology Inc., Boone, North Carolina, USA
- Storage: The strains are stored at -80 ± 10 °C in the Culture Collection of the Microbiological Laboratory of the testing facility. Frozen permanent cultures of the tester strains were prepared from fresh, overnight cultures to which DMSO was added as a cryoprotective agent.
- Confirmation of Phenotypes of Tester Strains: The phenotypes of the tester strains used in the bacterial reverse mutation assays with regard to membrane permeability (rfa), UV sensitivity (uvrA and uvrB), ampicillin resistance (amp), as well as spontaneous mutation frequencies are checked regularly according to Ames et al. and Maron and Ames.
-Spontaneous Reversion of Tester Strains: Each test strain reverts spontaneously at a frequency that is characteristic of the strain. Spontaneous reversion of the test strains to tryptophan independence is measured routinely in mutagenicity experiments and expressed as the number of spontaneous revertants per plate.
Historical control values for spontaneous revertants (revertants/plate) for untreated control sample without metabolic activation were in the period of 2011-2014 were as follows: Escherichia coli WP2 uvrA: 11-82.
- Procedure for Growing Cultures: The frozen bacterial cultures were thawed at room temperature and 200 μL inoculum were used to inoculate each 50 mL of Nutrient Broth No.2 for the overnight cultures in the assay. The cultures were incubated for 10-14 hours at 37 °C in a Gyrotory water bath shaker.
- Viability of the Testing Cultures: The viability of each testing culture was determined by plating 0.1 mL of the 10^5, 10^6, 10^7 and 10^8 dilutions prepared by sterile physiological saline on Nutrient Agar plates. The viable cell number of the cultures was determined by manual counting after approximately 24-hour incubation at 37 °C.
- Media: Nutrient Broth No.2 containing: Nutrient Broth No.2 25.0 g and Distilled water q.s. ad 1 000 mL. Sterilisation was performed at 121 °C in an autoclave.

Metabolic activation:

with and without

Metabolic activation system:

S9 Mix

Test concentrations with justification for top dose:

- Range Finding Test: 5 000, 2 500, 1 000, 316, 100, 31.6 and 10 μg/plate (Salmonella typhimurium TA98 and TA100 only)
- Initial Mutation Test and Confirmatory Mutation Test: Salmonella typhimurium strains were 1 581, 500, 158.1, 50, 15.81, 5, 1.581 and 0.5 μg/plate and Escherichia coli WP2 uvrA strain were 5 000, 1 581, 500, 158.1, 50, 15.81, 5 and 1.581 μg/plate.
- Concentrations were selected based on the results of the preliminary tests.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: DMSO was selected after a preliminary compatibility test was performed to examine the behaviour of the test material formulations with the solution of top agar and phosphate buffer.

PRELIMINARY COMPATABILITY TEST
-The solubility of the test material was examined using Distilled water, Dimethyl sulfoxide (DMSO) and Acetone. The test material was insoluble at 100 mg/mL concentration using Distilled water, however the formulation at the same concentration using DMSO (after approximately 3 minutes stirring) or Acetone were solutions and were suitable for the test. Due to the better biocompatibility DMSO (with the appropriate time of stirring) was selected as vehicle for the test.
The obtained stock solution (50 μL) with the solution of top agar and phosphate buffer was examined in a test tube without test bacterium suspension.

- Test solutions were freshly prepared at the beginning of the experiments in the testing laboratory by diluting the stock solution using the selected solvent.
- Analytical determination of the test material concentration, stability and homogeneity was not performed because of the character and the short period of study.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

methylmethanesulfonate

other: 4-nitro-1,2-phenylene-diamine (Without activation, Salmonella TA98 at 4 µg/plate) and 2-aminoanthracene (With activation, 2 µg/plate for all Salmonella strains and 50 µg/plate for E. coli WP2 uvrA)

Details on test system and experimental conditions:

METHOD OF APPLICATION: In the Range Finding Test as well as in the Initial Mutation Test, the plate incorporation method was used. In the Confirmatory Mutation Test, the pre-incubation method was used.

INITIAL MUTATION TEST
- A standard plate incorporation procedure was performed, as an Initial Mutation Test. Bacteria were exposed to the test material both in the presence and absence of an appropriate metabolic activation system.
- Molten top agar was prepared and kept at 45 °C. 2 mL of top agar was aliquoted into individual test tubes (3 tubes per control or concentration level). The equivalent number of minimal glucose agar plates was properly labelled. The test material and other components were prepared freshly and added to the overlay (45 °C).
- The content of the tubes: top agar 2 000 μL, vehicle or test material formulation (or reference controls) 50 μL, overnight culture of test strain 100 μL and phosphate buffer (pH 7.4) or S9 mix 500 μL. This solution was mixed and poured on the surface of minimal agar plates. For activation studies, instead of phosphate buffer, 0.5 mL of the S9 mix was added to each overlay tube. The entire test consisted of non-activated and activated test conditions, with the addition of untreated, negative (vehicle/solvent) and positive controls. After preparation, the plates were incubated at 37 °C for 48 hours.

CONFIRMATORY TEST
- A pre-incubation procedure was performed as a Confirmatory Mutation Test since in the Initial Mutation Test no positive effect was observed. Bacteria were exposed to the test material both in the presence and absence of an appropriate metabolic activation system. The equivalent number of minimal glucose agar plates was properly labelled. Molten top agar was prepared and kept at 45 °C.
- Before the overlaying, the test material formulation (or vehicle/solvent or reference control), the bacterial culture and the S9 mix or phosphate buffer (pH 7.4) was added into appropriate tubes to provide direct contact between bacteria and the test material (in its vehicle/solvent).
- The tubes (3 tubes per control or concentration level) were gently mixed and incubated for 20 min at 37 °C in a shaking incubator. After the incubation period, 2 mL of molten top agar was added to the tubes; the content was mixed up and poured onto minimal glucose agar plates as described for the standard plate incorporation method. The entire test consisted of non-activated and activated test conditions, with the addition of untreated, negative (vehicle/solvent) and positive controls. After preparation, the plates were incubated at 37 °C for 48 hours.

NUMBER OF REPLICATIONS: In the test each sample (including the controls) was tested in triplicate.

EVALUATION OF THE EXPERIMENTAL DATA
- The colony numbers on the untreated / negative (solvent) / positive control and test material treated plates were determined by manual counting. Visual examination of the plates was also performed; precipitation or signs of growth inhibition (if any) were recorded and reported.

Evaluation criteria:

CRITERIA FOR VALIDITY
The study was considered valid if:
- the number of revertant colonies of the negative (solvent) and positive controls were in the historical control range in all strains of the main tests
- at least five analysable concentrations were presented in all strains of the main tests.

CRITERIA FOR A POSITIVE RESPONSE:
A test material was considered mutagenic if:
- a dose–related increase in the number of revertants occurred and/or
- a reproducible biologically relevant positive response for at least one of the dose groups occurred in at least one strain with or without metabolic activation.
An increase was considered biologically relevant if:
- in all strains: the number of reversion was more than twice higher than the reversion rate of the negative (solvent) control.

CRITERIA FOR A NEGATIVE RESPONSE:
- A test material was considered non-mutagenic if it produced neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups, with or without metabolic activation.

Statistics:

The mean number of revertants per plate, the standard deviation and the mutation factor values were calculated for each concentration level of the test material and for the controls using Microsoft ExcelTM software.

Key result

Species / strain:

S. typhimurium, other: TA98, TA100, TA1535 and TA1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

PRELIMINARY RANGE FINDING TEST (INFORMATORY TOXICITY TEST)
- Inhibitory, cytotoxic effects of the test material (absent/reduced/slightly reduced background lawn development and/or reduced number of revertant colonies, in some cases pinpoint colonies were also detected) were observed in the Preliminary Range Finding Test in both tested strains without metabolic activation at 5 000, 2 500, 1 000 and 316 μg/plate concentrations and in both tested strains with metabolic activation at 5 000, 2 500 and 1 000 μg/plate concentrations.
- No precipitate of the test material was detected in the Preliminary Range Finding Test.

INITIAL AND CONFIRMATORY MUTATION TESTS
- In the Initial Mutation Test and Confirmatory Mutation Test none of the observed revertant colony numbers were above the respective biological threshold value. There were no reproducible dose-related trends and no indication of any treatment effect.
- In the Initial Mutation Test (plate incorporation method), the highest revertant rate was observed in Salmonella typhimurium TA1535 bacterial strain without metabolic activation at the concentration of 0.5 μg/plate. The mutation factor value was 1.91. Higher numbers of revertant colonies compared to the solvent control plates were observed at some other tested concentrations in this strain with metabolic activation. However, there was no dose-response relationship, the observed mutation factor values were below the biologically relevant threshold limit and the numbers of revertant colonies were within the historical control range.
- In the Confirmatory Mutation Test (pre-incubation method), the highest revertant rate was observed in Salmonella typhimurium TA1537 bacterial strain at 158.1 μg/plate concentration with metabolic activation. The mutation factor values were 1.80. However, there was no dose-response relationship, the observed mutation factor values were below the biologically relevant threshold limit and the numbers of revertant colonies were within the historical control range.
- Higher numbers of revertant colonies compared to the solvent control were detected in the Initial Mutation Test and Confirmatory Mutation Test in some other cases. However, no dose-dependence was observed and they were below the biologically relevant threshold value and were within the historical control range, they were considered as reflecting the biological variability of the test.
- Inhibitory, cytotoxic effects of the test material (absent/reduced/slightly reduced background lawn development and/or reduced number of revertant colonies, in some cases pinpoint colonies were also detected) were observed in the Initial Mutation Test at 1581 and 500 μg/plate concentrations in Salmonella typhimurium TA98, TA100, TA1537 strains with and without metabolic activation, in Salmonella typhimurium TA1535 strain without metabolic activation; at 1581 μg/plate concentration in Salmonella typhimurium TA1535 strain with metabolic activation and at 5 000 and 1 581 μg/plate concentrations in Escherichia coli WP2 uvrA strain with and without metabolic activation.
- Similar but stronger inhibitory, cytotoxic effects of the test material were observed in the Confirmatory Mutation Test in all Salmonella typhimurium bacterial strains at 1581, 500 and 158.1 μg/plate concentrations without metabolic activation and at 1 581 and 500 μg/plate concentrations with metabolic activation; in Escherichia coli WP2 uvrA strain without metabolic activation at 5 000, 1 581 and 500 μg/plate concentrations and at 5 000 and 1 581 μg/plate concentrations with metabolic activation.
- Slight precipitate was observed in the Confirmatory Mutation Test in Salmonella typhimurium strains with metabolic activation at the concentration of 1 581 and in Escherichia coli WP2 uvrA strain with metabolic activation at 5 000 and 1 581 μg/plate concentrations.

VALIDITY OF THE TESTS
- Untreated, negative (solvent) and positive controls were run concurrently. The mean values of revertant colony numbers of untreated, negative (solvent) and positive control plates were in good correlation with the historical control data. At least five analysable concentrations were presented in all strains of the main tests.
- The reference mutagens showed a distinct increase of induced revertant colonies. The viability of the bacterial cells was checked by a plating experiment in each test. The tests were considered to be valid.

Conclusions:

Under the conditions of this study, the test material had no mutagenic activity in the applied bacterium tester strains.

Executive summary:

The test material was tested for potential mutagenic activity in accordance with the standardised guidelines OECD 471, EU Method B13/14 and OPPTS 870.5100, under GLP conditions using the Bacterial Reverse Mutation Assay.

The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimurium TA98, TA100, TA1535 and TA1537) and the tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coli WP2 uvrA) in the presence and absence of a post mitochondrial supernatant (S9 fraction) prepared from the livers of phenobarbital/β-naphthoflavone-induced rats.

The study included a Preliminary Compatibility Test, a Preliminary Range Finding Test (Informatory Toxicity Test), an Initial Mutation Test (Plate Incorporation Method) and a Confirmatory Mutation Test (Pre-Incubation Method).

Based on the results of the Compatibility Test, the test material was dissolved in DMSO. Concentrations of 5 000, 2 500, 1 000, 316, 100, 31.6 and 10 μg/plate were examined in the Range Finding Test. Based on the results of the Range Finding Test, the test material concentrations in the Initial Mutation Test and Confirmatory Mutation Test for Salmonella typhimurium strains were 1 581, 500, 158.1, 50, 15.81, 5, 1.581 and 0.5 μg/plate and for Escherichia coli WP2 uvrA strain were 5 000, 1 581, 500, 158.1, 50, 15.81, 5 and 1.581 μg/plate.

In the Initial Mutation Test and Confirmatory Mutation Test none of the observed revertant colony numbers were above the respective biological threshold value. There were no reproducible dose-related trends and no indication of any treatment effect.

Inhibitory, cytotoxic effects of the test material were observed in the Initial Mutation Test at 1 581 and 500 μg/plate concentrations in Salmonella typhimurium TA98, TA100, TA1537 strains with and without metabolic activation, in Salmonella typhimurium TA1535 strain without metabolic activation; at 1581 μg/plate concentration in Salmonella typhimurium TA1535 strain with metabolic activation and at 5 000 and 1581 μg/plate concentrations in Escherichia coli WP2 uvrA strain with and without metabolic activation.

Similar but stronger inhibitory, cytotoxic effects of the test material were observed in the Confirmatory Mutation Test in all Salmonella typhimurium bacterial strains at 1 581, 500 and 158.1 μg/plate concentrations without metabolic activation and at 1 581 and 500 μg/plate concentrations with metabolic activation; in Escherichia coli WP2 uvrA strain without metabolic activation at 5 000, 1 581 and 500 μg/plate concentrations and at 5 000 and 1 581 μg/plate concentrations with metabolic activation.

Slight precipitate was observed in the Confirmatory Mutation Test in all tested strains with metabolic activation at the concentrations of 5 000 and/or 1 581 μg/plate.

The mean values of revertant colonies of the solvent control plates were in good correlation with the historical control data, the reference mutagens showed the expected increase in the number of revertant colonies, the viability of the bacterial cells was checked by a plating experiment in each test. At least five analysable concentrations were presented in all strains of the main tests. The tests were considered to be valid.

The reported data of this mutagenicity assay show that under the experimental conditions applied the test material did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Under the conditions of this study, the test material had no mutagenic activity in the applied bacterium tester strains.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 March 2019 to 09 April 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
The V79 cell line is well established in toxicology studies. Stability of karyotype and morphology makes it suitable for genetic toxicity assays with low background aberrations. These cells are chosen because of their small number of chromosomes (diploid number, 2n=22) and because of the high proliferation rates (doubling time 12 - 14 h). The V79 cell line was established after spontaneous transformation of cells isolated from the lung of a normal Chinese hamster (male).
The cell stocks were kept in a freezer at -80 ± 10 °C (for short-term storage) or in liquid nitrogen (long-term storage). The stock was checked for mycoplasma infection. No infection of mycoplasma was noted.
Trypsin-EDTA (0.25 % Trypsin, 1 mM EDTA) solution was used for cell detachment to subculture (cells were rinsed with 1X PBS before detachment). The laboratory cultures were maintained in 150 cm^2 plastic flasks at 37 ± 0.5 °C in a humidified atmosphere containing approximately 5 % CO2 in air. The V79 cells for this study were grown in Dulbecco’s Modified Eagle’s Medium supplemented with 2 mM L-glutamine, 1 % (v/v) Antibiotic-antimycotic solution (standard content: 10 000 NE/mL penicillin, 10 mg/mL streptomycin and 25 μg/mL amphotericin-B) and 10 % (v/v) heat-inactivated foetal bovine serum (DMEM-10, culture medium). When cells were growing well, subcultures were established in an appropriate number of flasks (after thawing, the cells were subcultured no more than 5 times before used in the study). During the treatments, the serum content of the medium was reduced to 5 % (v/v) (DMEM-5).

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system: S9 mix
- source of S9: In the experiments with metabolic activation in this study, a cofactor-supplemented post-mitochondrial S9 fraction prepared from activated rat liver was used as an appropriate metabolic activation system.
- method of preparation of S9 mix:
Induction of Rat Liver Enzymes: Male Wistar rats (292 - 387 g animals were 8 weeks old at the initiation) were treated with Phenobarbital (PB) and β-naphthoflavone (BNF) at 80 mg/kg/day by oral gavage for three consecutive days. Rats were given drinking water and food ad libitum until 12 hours before sacrifice when food was removed.

Preparation of Rat Liver Homogenate S9 Fraction: On Day 4, the rats were euthanised and the livers removed asceptically, weighed and washed several times in 0.15 M KCl. The washed livers were transferred to a beaker containing 3 mL of the 0.15 M KCl per g of wet liver and homogenised. Homogenates were centrifuged for 10 minutes at 9 000 g and the supernatant was decanted and retained. The freshly prepared S9 fraction was aliquoted into 1-5 mL portions, frozen, and stored at -80 ± 10 ºC.
The protein concentration of the preparation was determined by a chemical analyser at 540 nm. The protein concentration of the S9 fraction used in the study was determined to be 30.45 g/L. The sterility of the preparation was confirmed.
The biological activity in the Salmonella assay of S9 was characterised using the two mutagens 2 -aminoanthracene and benzo(a)pyrene, that requires metabolic activation by microsomal enzymes. The batch of S9 used in this study functioned appropriately.

Preparation of S9-mix: The complete S9-mix was freshly prepared on the day of use according to the following ratio:
S9 fraction: 3 mL
HEPES 20 mM: 2 mL
KCl 330 mM: 1 mL
MgCl2 50 mM: 1 mL
NADP 40 mM: 1 mL
Glucose-6-phosphate 50 mM: 1 mL
DME medium: 1 mL
Prior to addition to the culture medium the S9-mix was kept in an ice bath.
For all cultures treated in the presence of S9-mix, a 0.5 mL aliquot of the mix was added to each cell culture (final volume: 10 mL). The final concentration of the liver homogenate in the test system was 1.5 %.

Test concentrations with justification for top dose:

PRELIMINARY CYTOTOXICITY ASSAYS
- With and without metabolic activation: 0, 3.906, 7.813, 15.625, 31.25, 62.5, 125, 250, 500, 1 000, 2 000 µg/mL (3-hr treatment, harvesting 20-hr from the beginning of treatment)
- Without metabolic activation: 0, 3.906, 7.813, 15.625, 31.25, 62.5, 125, 250, 500, 1 000, 2 000 µg/mL (20-hr treatment, harvesting 20-hr from the beginning of treatment)

MAIN STUDY
Assay 1
- Without metabolic activation: 0, 7.5, 15, 25, 30, 40, 50 µg/mL (3-hr treatment, harvesting 20-hr from the beginning of treatment)
- With metabolic activation: 0, 7.5, 15, 25, 30, 40, 50 µg/mL (3-hr treatment, harvesting 20-hr from the beginning of treatment)

Assay 2
- Without metabolic activation: 0, 2.5, 5, 10, 15, 20, 25 µg/mL (20-hr treatment, harvesting 20-hr from the beginning of treatment)
- With metabolic activation: 0, 7.5, 15, 25, 30, 40, 50 µg/mL (3-hr treatment, harvesting 20-hr from the beginning of treatment)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Based on the result of the trial formulations of the test material, at 200 mg/mL concentration, insolubility was detected using distilled water, but powdered test material was soluble after approximately 2 minutes vortex using dimethyl sulfoxide (DMSO). Therefore, DMSO was selected for vehicle (solvent) of the study. The vehicle was compatible with the survival of the cells and the S9 activity.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Dimethyl sulfoxide (DMSO)

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Remarks:

Negative (vehicle) and positive control solutions were prepared immediately before the treatment of the cells and filtered sterile using a 0.22 μm syringe filter before use.

Details on test system and experimental conditions:

PRELIMINARY CYTOTOXICITY ASSAYS
Cells were treated for 3-hours in the presence and absence of S9-mix with a 20-hour harvesting time and for 20 hours in the absence of S9-mix with a 20-hour harvesting time.
The assays were performed with a range of test material concentrations to determine cytotoxicity. Treatment was performed as described for the main test. However, single cultures were used and positive controls were not included. Visual examination of the final culture medium was conducted at the beginning and end of the treatments. Measurement of pH and osmolality was also performed at the end of the treatment period.
At the scheduled harvesting time, the number of surviving cells was determined using a haemocytometer.

MAIN STUDY
- Treatment of the cells
For the cytogenetic experiments, 1-3 day old cultures (more than 50% confluency) were used. Cells were seeded into 92 x 17 mm tissue culture dishes at 5 x 10^5 cells/dish concentration and incubated for approximately 24 hours at 37°C in 10 mL of culture medium (DMEM-10). Duplicate cultures were used for each test material concentration or controls.
After the seeding period, the medium was replaced with 9.9 mL treatment medium (DMEM-5) in case of experiments without metabolic activation or with 9.4 mL treatment medium (DMEM-5) + 0.5 mL S9-mix in case of experiments with metabolic activation.
Cells were treated with different concentration test material solutions, untreated, negative (vehicle) or positive control solution (treatment volume was 100 μL/dish ) for the given period of time at 37°C in the absence or presence of S9-mix. After the exposure period, the cultures were washed with DMEM-0 medium (Dulbecco’s Modified Eagle’s Medium supplemented with 2 mM L-glutamine and 1% (v/v) Antibiotic-antimycotic solution). Then, 10 mL of fresh culture medium were added into the dishes and cells were incubated further until the scheduled harvesting time.
Harvesting was performed after 20 hours (approximately 1.5 normal cell cycles) from the beginning of treatment.
Solubility of the test material in the final treatment medium was visually examined at the beginning and end of the treatment in each case. Measurement of pH and osmolality was also performed at the end of the treatment period in the preliminary and both main tests.
For concurrent measurement of cytotoxicity an extra dish was plated for each sample and treated in the same manner. At the scheduled harvesting time, the number of surviving cells was determined using a haemocytometer.

- Preparation of Chromosomes
2-2.5 hours prior to harvesting, cell cultures were treated with Colchicine (0.2 μg/mL). The cells were swollen with 0.075 M KCl hypotonic solution for 4 minutes, then were washed in fixative (methanol : acetic-acid 3 : 1 (v : v) mixture) until the preparation became plasma free (4 washes). Then, a suspension of the fixed cells was dropped onto clean microscope slides and air-dried. The slides were stained with 5% Giemsa solution, air-dried and coverslips were mounted. At least three slides were prepared for each culture.

- Examination of Slides
Metaphase analysis was conducted for each test and the number of metaphases with aberrations (excluding gaps) and the types of aberrations for each culture were recorded. At least 150 metaphases with 22 ± 2 chromosomes (centromeres) from each culture (replicate) were examined for the presence or absence of chromosomal aberrations (approximately 1000x magnification), where possible. Chromatid and chromosome type aberrations (gaps, deletions and exchanges) were recorded separately.
When the origin of a fragment was clear, it was recorded under that category. When the origin of the fragment was not clear, it was recorded as a chromatid break. Metaphases with more than five aberrations (excluding gaps) were recorded as showing multiple damage. The examination of slides from a culture may be halted when 25 or more metaphases with aberrations (excluding gaps) have been recorded for that culture.
Additionally, the number of polyploid and endoreduplicated cells was scored. Marked reductions in the numbers of cells on the slides were also recorded.
The vernier co-ordinates of at least five metaphases (with aberrations, where possible) were recorded for each culture.

Results were expressed compared to the negative (vehicle) control as RICC% (Relative Increase in Cell Counts).

Evaluation criteria:

The assay is considered valid, if the following criteria are met:
- The negative (vehicle) control data are within the laboratory’s normal range for the spontaneous aberration frequency.
- The positive controls induce increases in the aberration frequency, which are significant.

The test material is considered to have shown clastogenic activity in this study if all of the following criteria are met:
- Increases in the frequency of metaphases with aberrant chromosomes are observed at one or more test concentrations (only data without gaps will be considered).
- The increases are reproducible between replicate cultures and between tests (when treatment conditions were the same).
- The increases are statistically significant.
- The increases are not associated with large changes in pH or osmolality of the treated cultures.
Evidence of a dose-response relationship (if any) was considered to support the conclusion.
The test material is concluded to have given a negative response if no reproducible, statistically significant increases are observed.

Statistics:

For statistical analysis, Fisher’s exact test was used. The parameter evaluated for statistical analysis was the number of cells with one or more chromosomal aberrations excluding gaps.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

MAIN STUDY
- Assay 1
Insolubility was not detected. There were no large changes in the pH and osmolality. Marked cytotoxicity was observed in the experiment with metabolic activation (RICC value of the highest evaluated concentration (40 μg/mL) with metabolic activation was 42 %). The same effect was observed in the experiment without metabolic activation (RICC value of the highest evaluated concentration (30 μg/mL) without metabolic activation was 41 %). Therefore, concentrations of 40, 15 and 7.5 μg/mL (a total of three) were chosen for evaluation in the experiment with metabolic activation, concentrations of 30, 15 and 7.5 μg/mL (a total of three) were chosen for evaluation in the experiment without metabolic activation.

- Assay 2
Insolubility was not detected. There were no large changes in the pH and osmolality. Marked cytotoxicity was observed in the experiment with metabolic activation (RICC value of the highest evaluated concentration (40 μg/mL) with metabolic activation was 45 %). The same effect was observed in the experiment without metabolic activation (RICC value of the highest evaluated concentration (20 μg/mL) without metabolic activation was 36 %). Therefore, concentrations of 40, 15 and 7.5 μg/mL (a total of three) were evaluated in the experiment with metabolic activation, and concentrations of 20, 10 and 5 μg/mL (a total of three) were evaluated in the experiment without metabolic activation.

None of the treatment concentrations caused a biologically or statistically significant increase in the number of cells with structural chromosome aberrations in either assay with or without metabolic activation when compared to the appropriate negative (vehicle) control values.
Polyploid metaphases and endoreduplicated metaphases were found in some cases in the negative (vehicle) control or positive control or test material treated samples in the performed experiments, but their incidence was not related to treatment with the test material.

In Assay 1 without metabolic activation and in Assay 2 with metabolic activation the mean% aberrant cells of the negative (vehicle) control was slightly higher than the maximum mean% aberrant cells of the general historical control range. This had no impact on the results or the integrity of the study since these differences were not significant.

VALIDITY OF THE STUDY
The tested concentrations in the chromosome aberration assays were selected based on the results of the preliminary experiments. Insolubility was not detected in all experiments with and without metabolic activation; while marked cytotoxicity was detected in Assay 1 and Assay 2 with and without metabolic activation. The evaluated concentration ranges of the Assays were considered to be adequate, as they covered the range from toxicity to no or little toxicity.
Three test material concentrations were evaluated in each experiment.
The spontaneous aberration frequencies of the negative (vehicle) controls in the performed experiments were within the acceptable range. (In the Assay 1 without metabolic activation and in Assay 2 with metabolic activation the mean% aberrant cells of the negative (vehicle) control was slightly higher than the maximum mean% aberrant cells of the general historical control range. This fact had no impact on the results or integrity of the study since these differences were not significant.)
In the performed experiments, the positive control substances (cyclophosphamide (CP) in the experiments with metabolic activation and ethyl methanesulfonate (EMS) in the experiments without metabolic activation) caused the expected statistically significant increase in the number of cells with structural chromosome aberrations.
The study was considered to be valid.

Summary of Results

Assay 1
Without Metaboilc Activation					With Metabolic Activation
Test Material	Conc (µg/mL) [No of analysed cells]	Time of Treatment/ Sampling	RICC† (%)	Mean % Aberrant Cells *	Test Material	Conc (µg/mL) [No of analysed cells]	Time of Treatment/ Sampling	RICC† (%)	Mean % Aberrant Cells *
Negative (vehicle) control	[300]	3 h / 20 h	100	3.3	Negative (vehicle) control	[300]	3 h / 20 h	100	2.3
Test material	0	3 h / 20 h	103	NE	Test material	0	3 h / 20 h	91	NE
	7.5 [300]	3 h / 20 h	93	1.7		7.5 [300]	3 h / 20 h	82	4.7
	15 [300]	3 h / 20 h	85	3.7		15 [300]	3 h / 20 h	63	3.3
	25	3 h / 20 h	71	NE		25	3 h / 20 h	63	NE
	30 [300]	3 h / 20 h	41	2.7		30	3 h / 20 h	59	NE
	40	3 h / 20 h	18	NE		40 [300]	3 h / 20 h	42	5.7
	50	3 h / 20 h	N/A	NE		50	3 h / 20 h	3	NE
Positive control	[247]	3 h / 20h	40	15.4**	Positive control	[60]	3 h / 20h	29	83.3**
Assay 2
Without Metaboilc Activation					With Metabolic Activation
Test Material	Conc (µg/mL) [No of analysed cells]	Time of Treatment/ Sampling	RICC† (%)	Mean % Aberrant Cells *	Test Material	Conc (µg/mL) [No of analysed cells]	Time of Treatment/ Sampling	RICC† (%)	Mean % Aberrant Cells *
Negative (vehicle) control	[300]	20 h/ 20 h	100	2.0	Negative (vehicle) control	[300]	3 h / 20 h	100	3.0
Test material	0	20 h/ 20 h	103	NE	Test material	0	3 h / 20 h	98	NE
	2.5	20 h/ 20 h	98	NE		7.5 [300]	3 h / 20 h	76	3.0
	5 [300]	20 h/ 20 h	87	1.3		15 [300]	3 h / 20 h	85	4.0
	10 [300]	20 h/ 20 h	73	0.7		25	3 h / 20 h	67	NE
	15	20 h/ 20 h	58	NE		30	3 h / 20 h	52	NE
	20 [300]	20 h/ 20 h	36	2.0		40 [300]	3 h / 20 h	45	3.0
	25	20 h/ 20 h	9	NE		50	3 h / 20 h	N/A	NE
Positive control	[279]	20 h/ 20h	39	17.9**	Positive control	[72]	3 h / 20h	52	69.4**

Negative (vehicle) control: DMSO

Positive control: (+S9) Cyclophosphamide, 6 μg/mL; (-S9) Ethyl methanesulfonate, 1 μL/mL (Assay 1), 0.4 μL/mL (Assay 2)

NE: Not evaluated
N/A: Not applicable

RICC: Relative Increase in Cell Counts

†: Compared to the negative (vehicle) control

*: Excluding gaps

**: p < 0.001 comparing numbers of aberrant cells excluding gaps with corresponding negative control.

Historical Control Data: 3/ 20 h Treatment/ Sampling Time Without S9-Mix

	Aberration Rate (Phases with Aberration in %)
	Negative Control		Positive Control (EMS)
	Incl. Gaps	Excl. Gaps	Incl. Gaps	Excl. Gaps
Mean	2.71	1.20	22.67	18.02
SD	1.65	0.82	12.43	8.16
Range	0 - 7	0 - 3	4 - 63	4 - 40
n	46	46	40	40

 

Historical Control Data: 3/ 20 h Treatment/ Sampling Time With S9-Mix

	Aberration Rate (Phases with Aberration in %)
	Negative Control		Positive Control (CP)
	Incl. Gaps	Excl. Gaps	Incl. Gaps	Excl. Gaps
Mean	3.24	1.45	73.00	69.50
SD	1.57	0.95	23.51	25.64
Range	0 - 8	0 - 4	21 - 100	21 - 100
n	43	43	21	21

SD = Standard deviation

Range = Min. – max. values

n = Number of experiments

EMS = Ethyl methanesulfonate

CP = Cyclophosphamide

In the period of 2008-2009, NNDA (N-Nitrosodimethylamine) was used as positive control substance in the experiments with metabolic activation. Mean aberration frequency for NNDA was 22.91 (including gaps) and 18.07 (excluding gaps) in 22 experiments.

In studies performed before the updated OECD guideline (2014) 200 metaphases were scored for chromosomal aberration per samples. Minimum and maximum values reflect the total number of aberrant cells in 200 metaphases. Furthermore, in those studies counting for a positive control sample was halted when 15 aberrant cells were counted.

Conclusions:

Under the conditions of the study the test material did not induce a significant level of chromosome aberrations in Chinese hamster V79 cells either with or without metabolic activation. Therefore, the test material was considered as not clastogenic in this test system.

Executive summary:

The clastogenic potential of the test material was investigated in vitro, in a study which was conducted in accordance with the standardised guideline OECD 473, under GLP condictions, using Chinese hamster V79 lung cells.

During the study the test material was formulated in DMSO and it was examined up to cytotoxic concentrations. In independent Chromosome Aberration Assays using duplicate cultures, at least 300 well-spread metaphase cells (or until a clear positive response was detected) were analysed for each evaluated test material treated, negative (vehicle) and positive control sample.

In Chromosome Aberration Assay 1, a 3-hour treatment with metabolic activation (in the presence of S9-mix) and a 3-hour treatment without metabolic activation (in the absence of S9-mix) were performed. Sampling was performed 20 hours after the beginning of the treatment in both cases. The examined concentrations of the test material were 50, 40, 30, 25, 15 and 7.5 μg/mL (experiment with and without metabolic activation).

In Assay 1, insolubility was not detected with and without metabolic activation. There were no large changes in the pH and osmolality. Marked cytotoxicity was observed in the experiment with metabolic activation (RICC value of the highest evaluated concentration (40 μg/mL) with metabolic activation was 42 %). The same effect was observed in the experiment without metabolic activation (RICC value of the highest evaluated concentration (30 μg/mL) without metabolic activation was 41 %). Therefore, concentrations of 40, 15 and 7.5 μg/mL (a total of three) were chosen for evaluation in the experiment with metabolic activation, concentrations of 30, 15 and 7.5 μg/mL (a total of three) were chosen for evaluation in the experiment without metabolic activation.

In Chromosome Aberration Assay 2, a 3-hour treatment with metabolic activation (in the presence of S9-mix) and a 20-hour treatment without metabolic activation (in the absence of S9-mix) were performed. Sampling was performed 20 hours after the beginning of the treatment in both cases. The examined concentrations of the test material were 50, 40, 30, 25, 15 and 7.5 μg/mL (experiment with metabolic activation) and 25, 20, 15, 10, 5 and 2.5 μg/mL (experiment without metabolic activation).

In Assay 2 insolubility was not detected with and without metabolic activation. There were no large changes in the pH and osmolality. Marked cytotoxicity was observed in the experiment with metabolic activation (RICC value of the highest evaluated concentration (40 μg/mL) with metabolic activation was 45 %). The same effect was observed in the experiment without metabolic activation (RICC value of the highest evaluated concentration (20 μg/mL) without metabolic activation was 36 %). Therefore, concentrations of 40, 15 and 7.5 μg/mL (a total of three) were evaluated in the experiment with metabolic activation, and concentrations of 20, 10 and 5 μg/mL (a total of three) were evaluated in the experiment without metabolic activation.

None of the treatment concentrations caused a biologically or statistically significant increase in the number of cells with structural chromosome aberrations in either assay with or without metabolic activation when compared to the appropriate negative (vehicle) control values.

Polyploid metaphases and/or endoreduplicated metaphases were found in some cases in the negative (vehicle) control or positive control or test material treated samples in the performed experiments, but their incidence was not related to treatment with the test material.

The negative (vehicle) control data were within the acceptable range for the spontaneous aberration frequency, the positive control substances caused a statistically significant increase in the number of structural aberrations excluding gaps in the experiments with or without metabolic activation demonstrating the sensitivity of the test system. The evaluated concentration range was considered to be adequate; three test material treated concentrations were evaluated in each assay. The tests were considered to be valid.

In conclusion, under the conditions of the study the test material did not induce a significant level of chromosome aberrations in Chinese hamster V79 cells either with or without metabolic activation. Therefore, the test material was considered as not clastogenic in this test system.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 August 2019 to 25 October 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Version / remarks:

29 July 2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

30 May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Specific details on test material used for the study:

No correction for purity was applied.

Target gene:

tk+/- (thymidine kinase) locus in L5178Y cells.

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
Cells were stored as frozen stocks in liquid nitrogen. Each batch of frozen cells was purged of TK-/- mutants and checked for the absence of mycoplasma. For each experiment, one or more vials was thawed rapidly, cells were diluted in RPMI-10 medium and incubated at 37 ± 0.5 °C in a humidified atmosphere containing approximately 5 % CO2 in air. When cells were growing well, subcultures were established in an appropriate number of flasks (after thawing, the cells were subcultured no more than four times before used in the main assays.

MEDIA USED
Three types of RPMI 1640 medium were prepared as follows:
- RPMI-5: containing 5 % v/v horse serum (heat inactivated), 0.01 mL/mL antibiotic-antimycotic solution, 0.5 mg/mL pluronic-F68 (Poloxamer 188), and 0.2 mg/mL pyruvic acid.
- RPMI-10: containing 10 % v/v horse serum (heat inactivated), 0.01 mL/mL antibiotic-antimycotic solution, 0.5 mg/mL pluronic-F68 (Poloxamer 188), and 0.2 mg/mL pyruvic acid.
- RPMI-20: containing 20 % v/v horse serum (heat inactivated), 0.01 mL/mL antibiotic-antimycotic solution, and 0.2 mg/mL pyruvic acid.
Heat inactivated horse serum was used in order to eliminate a factor which degrades TFT.
Standard content of Antibiotic-antimycotic solution: 10 000 IU/mL penicillin, 10 mg/mL streptomycin and 25 μg/mL amphotericin-B.
L-glutamine (0.3 mg/mL) or NaHCO3 was also added freshly to the media according to the manufacturer’s instruction.

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
The post-mitochondrial fraction (S9 fraction) was prepared from rat liver.
- Induction of Liver Enzymes: Male Wistar rats (292 - 387 g, 8 weeks old at initiation) were treated with Phenobarbital (PB) and β-naphthoflavone (BNF) at 80 mg/kg/day by oral gavage (for both inducers) for three consecutive days. Rats were given drinking water and food ad libitum until 12 hours before euthanasia when food was removed. Euthanasia was by ascending concentration of CO2, confirmed by cutting through major thoracic blood vessels. Initiation of the induction of liver enzymes used in the preparation of S9 fraction used in this study was 05 January 2018.
- Preparation of Rat Liver Homogenate S9 Fraction: On Day 4, the rats were euthanised and the livers removed aseptically using sterile surgical tools. After excision, livers were weighed and washed several times in 0.15 M KCl. The washed livers were transferred to a beaker containing 3 mL of 0.15 M KCl per g of wet liver, and homogenised. Homogenates were centrifuged for 10 minutes at 9 000 g and the supernatant was decanted and retained. The freshly prepared S9 fraction was distributed in 1 - 5 mL portions, frozen quickly and stored at -80 ± 10 °C. Sterility of the preparation was confirmed.
The protein concentration was determined by colorimetric test by chemical analyser at 540 nm. The protein concentration of the S9 fraction used was determined to be 30.45 g/L. The date of preparation of S9 fraction for this study was 08 January 2018.
The biological activity of each batch of S9 was characterised in the Salmonella assay using 2-Aminoanthracene and Benzo(a)pyrene, that requires metabolic activation by microsomal enzymes. The batch of S9 used in this study was found active under the test conditions.

The S9-mix (sterile) used for the treatments was prepared as follows:
0.2 mL/mL potassium chloride (150 mM), 0.2 mL/mL NADP (sodium salt) (25 mg/mL), 0.2 mL/mL D-Glucose-6-phosphate (monosodium salt) (180 mg/mL), 0.4 mL/mL S9 fraction.

For all cultures treated in the presence of S9-mix, a 1 mL aliquot of the mix was added to each cell culture (19 mL) to give a total of 20 mL. The final concentration of the liver homogenate in the test system was 2 %. Cultures treated in the absence of S9-mix received 1 mL of 150 mM KCl (except for the 24-hour treatment). Prior to addition to the culture medium, the S9-mix was kept in an ice bath.

Test concentrations with justification for top dose:

The concentrations used in the main tests were as follows:
Assay 1 (treatment period: 3 hours): 0, 10, 20, 40, 60, 70, 80, 90, 100 µg/mL (with and without metabolic activation)
Assay 2 (treatment period: 3 hours): 0, 10, 20, 40, 60, 70, 80, 90, 100 µg/mL (with metabolic activation)
Assay 2 (treatment period: 24 hours): 0, 5, 10, 20, 30, 40, 45, 50, 55 µg/mL (without metabolic activation)

Treatment concentrations for the mutation assays were selected on the basis of the result of a short preliminary toxicity test. Three-hour treatment in the presence and absence of S9-mix and 24-hour treatment in the absence of S9-mix was performed with a range of test material concentrations to determine toxicity immediately after the treatments.
The highest concentration tested in the preliminary test was 2 000 μg/mL (the recommended maximum concentration). Treatment of cell cultures was made as described in the next section for the main mutation assays. However, single cultures were only used and positive controls were not included. After the treatment period, cell concentrations were determined using a haemocytometer. Cells were transferred for the expression period for two extra days and repeated cell counting was performed. Visual examination for precipitation of test material in the final culture medium was conducted at the beginning and end of the treatments. Measurement of pH and osmolality was also performed after the treatment period.
Insolubility and cytotoxicity were detected in the preliminary experiments. The concentrations were selected for the main assays according to the OECD No. 490 guideline instructions. Eight concentrations were selected for the main tests.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: the vehicle was selected based on information available from a previous study in which the test material was found to be soluble at 200 mg/mL concentration using DMSO as vehicle. This vehicle (solvent) was compatible with the survival of the cells and the S9 activity.

For the treatments in the study, stock formulations (200 mg/mL was used in the preliminary experiment and 100 mg/mL was used in the main assays) were prepared in the testing laboratory as follows. The necessary amount of the test material was weighed into a calibrated volumetric flask; approximately 80 % of the required volume of the vehicle (solvent) was added and stirred by a vortex until homogeneity was reached. Then, it was filled up to the final volume with the vehicle (solvent) to form a stock formulation. From the stock formulation several dilutions were prepared using the selected vehicle for dosing formulations. In each case, the stock formulation and the vehicle were filtered sterile using a 0.22 μm syringe filter before the preparation of the dosing formulations. The stock formulations and all the dilutions were prepared immediately before the treatment of the cells in a sterile hood.
Analytical determination of the test material concentration, stability and homogeneity was not performed because of the character and the short period of study.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

cyclophosphamide

Details on test system and experimental conditions:

MAIN MUTATION ASSAYS
In Assay 1, cells were treated for 3 hours in the presence and absence of S9-mix. In Assay 2, cells were treated for 3 hours in the presence of S9-mix and for 24 hours in the absence of S9-mix.
- Eight test material concentrations were plated for mutagenicity testing in each case for Assay 1.
- At least six test material concentrations were plated for mutagenicity testing in each case for Assay 2.
A suitable volume (0.2 mL for a final volume of 20 mL (10 μL/mL) of RPMI-5 medium, vehicle (solvent), test material formulations or positive control solutions, and 1.0 mL of S9-mix (in experiments with metabolic activation) or 1.0 mL of 150 mM KCl (in case of 3-hour treatment without metabolic activation) were added to a final volume of 20 mL per culture in each experiment. For the 3-hour treatments, 10^7 cells were placed in each of a series of 75 cm^2 sterile flasks. For the 24-hour treatment, 6 x 10^6 cells were placed in each of a series of 25 cm^2 sterile flasks. The treatment medium contained a reduced serum level of 5 % (v/v) RPMI-5.
Duplicate cultures were used for each treatment. Cultures were visually examined at the beginning and end of treatments. During the treatment period, cultures were incubated at 37 ± 1 °C (approximately 5 % CO2 in air). Gentle shaking was used during the treatments. Measurement of pH and osmolality was also performed after the treatment period.
Then cultures were centrifuged at 2 000 rpm (approximately 836 g) for 5 minutes, washed with tissue culture medium and suspended in at least 20 mL RPMI-10. The number of viable cells in the individual samples was counted manually using a haemocytometer. Where sufficient cells survived, cell density was adjusted to a concentration of 2 x 10^5 cells/mL (if possible). Cells were transferred to flasks for growth through the expression period (maximum 30 mL of suspension) or diluted to be plated for survival.

PLATING FOR SURVIVAL
Cultures of cell density 2 x 10^5 cells/mL, were further diluted to 8 cells/mL. Using a multi-channel pipette, 0.2 mL of the final concentration of each culture were placed into each well of two, 96-well microplates (192 wells) averaging 1.6 cells per well. Microplates were incubated at 37 ± 0.5 °C containing approximately 5 % (v/v) CO2 in air for two weeks. Wells containing viable clones were identified by eye using background illumination and counted.

EXPRESSION PERIOD
To allow expression of TK- mutations, cultures were maintained in flasks for 2 days. During the expression period, subculturing was performed daily. On each day, cell density was adjusted to a concentration of 2 x 10^5 cells/mL (whenever possible) and transferred to flasks for further growth.
On completion of the expression period, at five test material treated samples, untreated, negative (vehicle) and positive controls were plated for determination of viability and 5-trifluorothymidine (TFT) resistance.

PLATING FOR VIABILITY
At the end of the expression period, the cell density in the selected cultures was determined and adjusted to 1x10^4 cells/mL with RPMI-20 for plating for a viability test. Using a multi-channel pipette, 0.2 mL of the final concentration of each culture was placed into each well of two, 96-well microplates (192 wells) averaging 1.6 cells per well. Microplates were incubated at 37 ± 0.5 °C containing approximately 5 % (v/v) CO2 in air for approximately two weeks (12 days). Wells containing viable clones were identified by eye using background illumination and counted.

PLATING FOR TFT RESISTANCE
At the end of the expression period, the cell concentration was adjusted to 1 x 10^4 cells/mL. TFT (300 μg/mL stock solution) was diluted 100-fold into these suspensions to give a final concentration of 3 μg/mL. Using a multi-channel pipette, 0.2 mL of each suspension was placed into each well of four, 96-well microplates (384 wells) at 2 x 10^3 cells per well.
Microplates were incubated at 37 ± 0.5 °C containing approximately 5 % (v/v) CO2 in air for approximately two weeks (12 days) and wells containing clones were identified by eye and counted. In addition, scoring of large and small colonies was performed to obtain information on the possible mechanism of action of the test material, if any.

ANALYSIS OF THE RESULTS
- Determination of Survival or Viability
From the zero term of the Poisson distribution the probable number of clones/well (P) on microplates in which there are empty wells (EW, without clones) out of a total of wells (TW) is given by:

P = -ln (EW/TW)

The plating efficiency (either after the treatment = PEsurvival or at the end of the expression period = PEviability) in any given culture is therefore:

PE = P/1.6
The percentage relative survival (%RS) in each test culture was therefore determined by comparing plating efficiencies (PEsurvival) in test and control cultures thus:

%RS = [PE (test)/PE (control)] x 100

To take into account any loss of cells during the treatment period, percentage relative survival value for each dose of test item was adjusted as follows:

Adjusted %RS = % RS x (Post treatment cell concentration for dose / Post treatment cell concentration for solvent control)

All percentage relative survival (%RS) values were adjusted as described above.

- Calculation of Suspension Growth (SG) and Relative Total Growth (RTG)
The RTG was then calculated as the percentage total growth of the treated cultures compared to the corresponding negative (vehicle/solvent) control value.
Suspension growth one (SG1) is the growth rate between Day 0 and Day 1 (cell concentration at Day 1 / cell concentration at Day 0) and Suspension growth two (SG2) is the growth rate between Day 1 and Day 2 (cell concentration at Day 2 / cell concentration at Day 1). The Relative Suspension Growth (RSG) is the total SG (SG1 x SG2) for the treated culture compared to the vehicle (solvent) control. In case of the long treatment (24-hour) without metabolic activation, the day of treatment was similarly taken into consideration during calculation.

RSG = [SG1(test) x SG2(test)] / [SG1(control) x SG2(control)]

Relative Cloning Efficiency (RCE) is the relative cloning efficiency of the test culture compared to the relative cloning efficiency of the negative (vehicle) control obtained at the time of mutant selection.
The Relative Total Growth (RTG) was calculated as follows:

RTG = RSG x RCE
The RTG was given as the percentage value of the treated cultures compared to the corresponding negative (vehicle/solvent) control value.

- Determination of Mutant Frequency
It is usual to express mutant frequency (MF) as "mutants per 10^6 viable cells". In order to calculate this, the plating efficiencies of both mutant and viable cells in the same culture were calculated:

MF = [P (mutant)/2 x 10^3] x [1.6/P (viable)] x 10^6
= {-ln [EW/TW (mutant)]/-ln [EW/TW (viable)]} x 800

Small and large colony mutant frequencies can be calculated in an identical manner, using the relevant number of empty wells for small and large colonies as appropriate.

Evaluation criteria:

The test material was considered to be clearly positive (mutagenic) in this assay if all the following criteria were met:
1. At least one concentration exhibited a statistically significant increase (p < 0.05) compared with the concurrent negative (vehicle) control and the increase was biologically relevant (i.e. the mutation frequency at the test concentration showing the largest increase was at least 126 mutants per 10^6 viable cells (GEF = the Global Evaluation Factor) higher than the corresponding negative (vehicle/solvent) control value).
2. The increases in mutation frequency were reproducible between replicate cultures and/or between tests (under the same treatment conditions).
3. The increase was concentration-related (p < 0.05) as indicated by the linear trend analysis.
The test material was considered clearly negative (non-mutagenic) in this assay if in all experimental conditions examined there was no concentration related response or, if there is an increase in MF, but it did not exceed the GEF. Then, test material was considered unable to induce mutations in this test system.
Results, which only partially satisfied the acceptance and evaluation criteria, were evaluated on a case-by-case basis. Similarly, positive responses seen only at high levels of cytotoxicity required careful interpretation when assessing their biological significance. Caution was exercised with positive results obtained at levels of cytotoxicity lower than 10 % (as measured by RTG).

Statistics:

Statistical significance of mutant frequencies (total wells with clones) was performed using Microsoft Excel software.
The negative (vehicle/solvent) control log mutant frequency (LMF) was compared to the LMF of each treatment concentration, based on Dunnett's test for multiple comparisons and the data were checked for a linear trend in mutant frequency with treatment dose using weighted regression. The test for linear trend was one-tailed, therefore negative trend was not considered significant. These tests required the calculation of the heterogeneity factor to obtain a modified estimate of variance.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

PRELIMINARY EXPERIMENT
In the preliminary experiment cytotoxicity and insolubility were detected with and without metabolic activation.

MUTATION ASSAYS
> Assay 1
In Assay 1, there were no large changes in pH or osmolality after treatment with or without metabolic activation. No insolubility was observed in the final treatment medium at the end of the treatment with or without metabolic activation.
- Presence of S9-mix (3-hour treatment)
In the presence of S9-mix (3-hour treatment), cytotoxicity was seen at concentration range of 60 - 100 μg/mL. The 100 and 90 μg/mL concentrations showed marked cytotoxicity (relative total growth (RTG): 3 % and 9 %, respectively) and they were excluded from the evaluated concentration range. Thus, an evaluation was made using data of six concentrations (concentration range of 10 - 80 μg/mL). Relative total growth of the highest evaluated concentration (80 μg/mL) showed proper degree of cytotoxicity (RTG was 19 %).
There was statistically significant increase in the mutation frequency value of the four highest evaluated concentrations (40 - 80 μg/mL), the difference between the calculated values and the control exceed the Global Evaluation Factor, GEF (thus showing biological relevance) in the concentrations of 80, 70 and 60 μg/mL. At 40 μg/mL and lower concentrations the mutation frequency value was below the GEF. A concentration related increase was indicated by the linear trend analysis.
This experiment was considered as being a positive.
- Absence of S9-mix (3-hour treatment)
In the absence of S9-mix (3-hour treatment), cytotoxicity of the test material was observed at higher concentrations (concentration range of 70 - 100 μg/mL), lower degree of cytotoxicity was observed at 60 μg/mL (RTG value was 66 %) and no significant cytotoxicity was observed at lower concentrations. An evaluation was made using data of eight concentrations (concentration range of 10 - 100 μg/mL). Relative total growth of the highest evaluated and tested concentration (100 μg/mL) was 26 %. No statistically significant or biologically relevant increase in the mutation frequency was noted at any of the evaluated concentrations. Concentration related increase was indicated by the linear trend analysis. However, based on the individual values the difference between the calculated values and the control did not exceed the Global Evaluation Factor, GEF, thus it was not biologically relevant. This experiment was considered as being negative.

> Assay 2
In Assay 2, there were no large changes in pH or osmolality after treatment with or without metabolic activation. No insolubility was observed in the final treatment medium at the end of the treatment with or without metabolic activation.
- Presence of S9-mix (3-hour treatment)
In the presence of S9-mix (3-hour treatment), cytotoxicity was seen at concentration range of 60 - 100 μg/mL, lower degree of cytotoxicity was observed at 40 μg/mL (RTG value was 61 %) and no significant cytotoxicity was observed at lower concentrations.
An evaluation was made using data of eight concentrations (concentration range of 10 - 100 μg/mL). Relative total growth of the highest evaluated and tested concentration (100 μg/mL) was 36 %).
There was statistically significant increase in the mutation frequency value of the six highest evaluated concentrations (40 - 100 μg/mL), the difference between the calculated values and the control exceed the Global Evaluation Factor, GEF (thus showing biological relevance) in the concentrations of 100, 90, 80, 70 and 60 μg/mL. At 40 μg/mL or lower concentrations the mutation frequency value was below the GEF. A concentration related increase was indicated by the linear trend analysis.
Lower degree of cytotoxicity was observed at the three highest concentrations in Assay 2 compared to Assay 1, however reproducibility was observed at the evaluated concentrations and the effect was reproducible between assays. Therefore, this experiment was considered to be reproducibly positive.
- Absence of S9-mix (24-hour treatment)
In the absence of S9-mix (24-hour treatment), cytotoxicity of the test material was observed at concentration range of 30 - 55 μg/mL. No cells survived the expression period in the samples of 55 and 50 μg/mL concentrations and excessive cytotoxicity was observed at 45 and 40 μg/mL concentrations. An evaluation was made using data of five concentrations (concentration range of 5 - 40 μg/mL). Relative total growth of the highest evaluated concentration (40 μg/mL was 1 %) and the RTG value of the next evaluated concentration (30 μg/mL) was 25 %, thus the range was covered between 1 % and 25 %. Although the cytotoxicity at 40 μg/mL was excessive, it was considered to be useful to include within the data evaluation. There was statistically significant increase in the mutation frequency values in the highest evaluated concentration in Assay 2 and concentration related increase was also indicated by the linear trend analysis. Based on the individual values the difference between the calculated values and the control exceed the Global Evaluation Factor, GEF, thus it was biologically relevant. However, this effect was seen only at the highest evaluated dose (which was used for evaluation for cytotoxicity data interpretation) at an excessive cytotoxicity level, therefore this result would not be considered positive. Furthermore, if this concentration (40 μg/mL) is excluded from the evaluation no statistically significant or biologically relevant increase in the mutation frequency was observed and no concentration related increase was indicated by the linear trend analysis. This experiment was considered as being negative.

VALIDITY OF THE MUTATION ASSAYS
- Untreated, negative (vehicle/solvent) and positive controls were run concurrently in the study. The spontaneous mutation frequency of the negative (vehicle/solvent) and untreated controls were in the recommended range (50 - 170 x 10^-6) in all cases.
- The positive controls (Cyclophosphamide in the presence of metabolic activation and 4-Nitroquinoline-N-oxide in the absence of metabolic activation) gave the anticipated increases in mutation frequency over the controls and were in accordance with historical data in all assays. All of the positive control samples in the performed experiments fulfilled at least one of the relevant OECD No. 490 criteria.
- The plating efficiencies for the negative (vehicle/solvent) controls of the test material and positive control item as well as the untreated control samples at the end of the expression period (PEviability) were acceptable in all assays.
Slightly higher value of the negative (vehicle/solvent) control (123.8 %) than the upper limit of the suggested range (65 - 120%) was detected in Assay 2 with metabolic activation, but as the mutation frequency value of this control was in the acceptable range, this fact was considered not to adversely affect the results of the study, and the observed value was acceptable.
Slightly higher value of the untreated control (138.3 %, 139.8 % and 147.9 %) than the upper limit of the suggested range (65 – 120 %) was detected in Assay 1 without metabolic activation and in Assay 2 with or without metabolic activation, but as the mutation frequency value of this control was in the acceptable range, this fact was considered not to adversely affect the results of the study, and the observed value was acceptable.
- The number of test concentrations evaluated was at least five in Assay 1 and Assay 2, which met the acceptance criteria about the minimum number of evaluated concentrations.
- The tested concentration range in the study was considered to be adequate as the highest evaluated concentration showed proper degree of cytotoxicity (approximately 80 - 90 %, i.e. approximately 10 - 20 relative total growth). Lower test concentrations were usually spaced by a factor of two, but more closely spaced concentration were used in the expected cytotoxic range in all cases in an attempt to obtain values in the range of 10 - 20 % viability.
In Assay 2 with metabolic activation the relative total growth of the highest evaluated concentration (100 μg/mL) was 36 %, which showed positive response and it was slightly above the target range according to the OECD guideline, however in Assay 1 higher degree of cytotoxicity was observed at this concentration and the highest evaluated concentration (80 μg/mL) showed positive response at proper degree of cytotoxicity (RTG was 19 %). The positive response was reproducible between assays. Thus, the results were considered to cover appropriate concentrations, being acceptable to justify the study for the exposures with metabolic activation.
In Assay 1 without metabolic activation the relative total growth of the highest evaluated concentration (100 μg/mL) was 26 %, and in Assay 2 without metabolic activation the relative total growth of the highest evaluated concentration (40 μg/mL) was 1 % and the next evaluated concentration (30 μg/mL) was 25 % (a very steep dose response). A lower or higher degree of cytotoxicity was observed in each case than the recommendation of the relevant OECD guideline.
However, in Assay 2 no cells survived at 55 and 50 μg/mL or excessive cytotoxicity was observed at 45 μg/mL concentrations. A closely spaced concentration range was used to properly cover concentrations from cytotoxicity to no cytotoxicity, and the results were considered to be negative in both short and long treatment without metabolic activation. Therefore, it is concluded that it is valid to state that the test item is negative under this condition without metabolic activation. In addition, it had no effect on the final conclusion with metabolic activation, which was positive.
- Suspension growth value of the negative (vehicle) control were lower than the recommended value in Assay 2, however as all of the observed spontaneous mutation frequency values of the negative (vehicle) control samples were in the recommended range, this fact was considered to be acceptable and not to adversely affect the results of the study. Further suspension growth value of the untreated and negative (vehicle/solvent) control samples were in line with the recommended range.
The overall study was considered to be valid.

Summary of Mutagenicity Data (Assay 1)

Test Material or Control Conc.	Number of Empty Wells/Total Number of Wells	Number of Large Colonies/Total Number of Wells	Number of Small Colonies/ Total Number of Wells	Dn²/var(Dn)†	Mutation Frequency	Increase in MF Compared to Vehicle Control (GEF)
3 Hour Treatment Period With Metabolic Actvation
100 µg/mL	NE	NE	NE	NE	NE	NE
90 µg/mL	NE	NE	NE	NE	NE	NE
80 µg/mL	478/768	125/768	165/768	30.456*	307.9	211.8 (>GEF)
70 µg/mL	484/768	121/768	163/768	19.047*	241.1	145.0 (>GEF)
60 µg/mL	473/768	131/768	164/768	19.226*	241.3	145.2 (>GEF)
40 µg/mL	561/768	86/768	121/768	6.696*	169.2	73.1 (<GEF)
20 µg/mL	610/768	72/768	86/768	0.843	118.4	22.3 (<GEF)
10 µg/mL	655/768	48/768	65/768	0.193	86.4	-9.7 (<GEF)
Vehicle control	638/768	63/768	67/768	--	96.1	--
Untreated control	639/768	63/768	66/768	--	92.3	--
Positive control (CP: 4 μg/mL)	90/768	309/768	369/768	‡3.03E^-17	2089.8*	--
3 Hour Treatment Period Without Metabolic Actvation
100 µg/mL	533/768	84/768	151/768	4.413	150.4	55.7 (<GEF)
90 µg/mL	559/768	88/768	121/768	2.860	138.1	43.4 (<GEF)
80 µg/mL	611/768	67/768	90/768	0.743	115.7	21.0 (<GEF)
70 µg/mL	626/768	67/768	75/768	0.140	103.4	8.7 (<GEF)
60 µg/mL	633/768	71/768	64/768	0.098	87.8	-6.9 (<GEF)
40 µg/mL	652/768	57/768	59/768	0.170	85.5	-9.2 (<GEF)
20 µg/mL	663/768	44/768	61/768	0.164	85.5	-9.2 (<GEF)
10 µg/mL	649/768	64/768	55/768	0.111	87.2	-7.5 (<GEF)
Vehicle control	630/768	69/768	69/768	--	94.7	--
Untreated control	620/768	82/768	66/768	--	77.4	--
Positive control (NQO: 0.15 μg/mL)	182/768	274/768	312/768	‡9.4E^-13	1149.7*	--

* Statistically significant
† Evaluated by Dunnett’s test for multiple comparisons.
‡ Evaluated by T-test for independent samples (compared to the DMSO vehicle control).
Dn: Difference of log mutant frequency of dose “n” and that of the vehicle control
var(Dn): variance of Dn β = slope of the curve, var(β) = variance of the slope
Negative (vehicle) control: DMSO
DMSO: Dimethyl sulfoxide
CP: Cyclophosphamide
NQO: 4-Nitroquinoline-N-oxide
GEF: Global Evaluation Factor (=126 per 10^6 viable cells)
Note: Mutation frequency refers to 10^6 viable cells
NE: Not evaluated due to the cytotoxicity

With S9-mix: In linear trend analysis β2/var (β) = 57.28, significant at p<0.001

Without S9-mix: In linear trend analysis β2/var (β) = 7.41, significant at p<0.01

Summary of Mutagenicity Data (Assay 2)

Test Material or Control Conc.	Number of Empty Wells/Total Number of Wells	Number of Large Colonies/Total Number of Wells	Number of Small Colonies/ Total Number of Wells	Dn²/var(Dn)†	Mutation Frequency	Increase in MF Compared to Vehicle Control (GEF)
3 hour treatment period with metabolic actvation
100 µg/mL	362/768	155/768	251/768	34.922*	303.8	217.3 (>GEF)
90 µg/mL	398/768	156/768	214/768	34.096*	301.2	214.7 (>GEF)
80 µg/mL	444/768	154/768	170/768	23.504*	246.8	160.3 (>GEF)
70 µg/mL	449/768	140/768	179/768	23.675*	248.1	161.6 (>GEF)
60 µg/mL	445/768	157/768	166/768	20.573*	231.0	144.5 (>GEF)
40 µg/mL	518/768	121/768	129/768	6.775*	154.5	68.0 (<GEF)
20 µg/mL	561/768	94/768	113/768	2.960	128.1	41.6 (<GEF)
10 µg/mL	602/768	83/768	83/768	0.298	98.4	11.9 (<GEF)
Vehicle control	620/768	73/768	75/768	--	86.5	--
Untreated control	612/768	82/768	76/768	--	81.2	--
Positive control (CP: 4 μg/mL)	95/768	311/768	362/768	‡1.4^-17	1610.7*	--
Test Material or Control Conc.	Number of Empty Wells/Total Number of Wells	Number of Large Colonies/Total Number of Wells	Number of Small Colonies/ Total Number of Wells	Dn²/var(Dn)	Mutation Frequency	Increase in MF Compared to Vehicle Control (GEF)
24 hour treatment period withOUT metabolic actvation
55 µg/mL	ND	ND	ND	ND	ND	ND
50 µg/mL	ND	ND	ND	ND	ND	ND
45 µg/mL	NE	NE	NE	NE	NE	NE
40# µg/mL	566/768	84/768	118/768	20.158*	247.2#	160.2# (>GEF)
30 µg/mL	601/768	84/768	83/768	1.759	119.1	32.1 (<GEF)
20 µg/mL	610/768	81/768	77/768	0.079	93.0	6 (<GEF)
10 µg/mL	604/768	86/768	78/768	0.057	82.1	-4.9 (<GEF)
5 µg/mL	658/768	55/768	55/768	0.147	78.8	-8.2 (<GEF)
Vehicle control	631/768	68/768	69/768	--	87.0	--
Untreated control	601/768	94/768	73/768	--	82.9	--
Positive control (NQO: 0.1 μg/mL)	125/768	346/768	297/768	‡ 1.25^-11	1647.2*	--

* Statistically significant
† Evaluated by Dunnett’s test for multiple comparisons.
‡ Evaluated by T-test for independent samples (compared to the DMSO vehicle control).
Dn: Difference of log mutant frequency of dose “n” and that of the vehicle control
var(Dn): variance of Dn β = slope of the curve var(β) = variance of the slope
Negative (vehicle) control: DMSO
DMSO: Dimethyl sulfoxide
CP: Cyclophosphamide
NQO: 4-Nitroquinoline-N-oxide
GEF: Global Evaluation Factor (=126 per 10^6 viable cells)
Note: Mutation frequency refers to 10^6 viable cells
NE: Not evaluated due to the cytotoxicity
ND: No data (Cells did not survive the treatment or expression period.)
# Excessive cytotoxicity was observed at this concentration; however, it was used for the evaluation for cytotoxicity data interpretation purposes. The mutation frequency of this concentration was above the GEF and showed statistically significant result, but the increase in MF occurred only at excessive cytotoxicity, thus the result would not be considered positive.

With S9-mix: In linear trend analysis β2/var (β) = 83.43, significant (at p<0.001)

Without S9-mix: In linear trend analysis β2/var (β) = 10.17, significant (at p<0.001)

Historical Control Data: Mutation Frequency of the Negative Controls (2006 – 2016)

Treatments	Culture Medium			Distilled Water			DMSO
	3 h +S9	3 h – S9	24 h -S9	3 h +S9	3 h – S9	24 h -S9	3 h +S9	3 h – S9	24 h -S9
Average	94.3	103.6	106.4	90.4	96.6	96.3	97.3	97.3	98.9
SD	26.9	35.3	27.4	22.7	19.0	24.6	33.7	38.5	26.8
Min.	39.3	52.6	41.7	33.4	55.1	43.2	44.2	33.7	47.1
Max.	198.5	235.6	179.1	121.8	125.0	141.1	269.9	261.6	159.4
n	84	43	44	26	13	13	101	57	50

 

Historical Control Data: Mutation Frequency of the Positive Controls (2006 – 2016)

Treatments	Cyclophosphamide	4-Nitroquinoline-N-oxide
	3 h + S9	3 h -S9	24 h -S9
Average	1178.7	722.2	831.9
SD	524.7	330.0	337.2
Min.	196.1	223.5	245.0
Max.	2642.5	1687.3	1577.6
n	106	58	52

h: Hour

SD: Standard Deviation

+S9: Experiment with metabolic activation

-S9: Experiment without metabolic activation

n: Number of cases

Conclusions:

Under the conditions of the study, treatment with the test material did result in a statistically significant and biologically relevant, dose dependent increase in the mutation frequency in the presence of a rat metabolic activation system (S9 fraction) in Assay 1, the observed effect was repeatable within or between assays. Therefore, reproducible positive effect of the test material was concluded in the performed experiments; overall the test material was considered to be positive with metabolic activation.
Negative results were seen in the experiment in the absence of a rat metabolic activation system (S9 fraction). Statistical differences were not supported by any results above the GEF with the exception of one concentration in Assay 2 without metabolic activation, however in this case excessive cytotoxicity was observed, thus the result would not be considered positive.
The Mouse Lymphoma Assay was considered to be valid and to reflect the real potential of the test material to cause mutations in the cultured mouse cells used in this study.
Overall the test material was considered to be mutagenic.

Executive summary:

An in vitro mammalian cell assay was performed in mouse lymphoma L5178Y TK+/- 3.7.2 C cells at the tk locus to test the potential of the test material to cause gene mutation and/or chromosome damage. The study was conducted in accordance with the standardised guidelines OECD 490 and EU Method B.17, under GLP conditions.

Treatment was performed for 3 hours with and without metabolic activation (±S9 mix) and for 24 hours without metabolic activation (-S9 mix).

Dimethyl sulfoxide (DMSO) was used as vehicle of the test material in this study. Based on the preliminary toxicity test, the following test material concentrations were examined in the mutation assays:

Assay 1, 3-hour treatment with and without metabolic activation: 100, 90, 80, 70, 60, 40, 20 and 10 μg/mL,

Assay 2, 3-hour treatment with metabolic activation: 100, 90, 80, 70, 60, 40, 20 and 10 μg/mL,

Assay 2, 24-hour treatment without metabolic activation: 55, 50, 45, 40, 30, 20, 10 and 5 μg/mL.

In Assays 1-2 with and without metabolic activation, there were no large changes in pH or osmolality after treatment. No insolubility was observed in the final treatment medium at the end of the treatment in Assays 1-2 with and without metabolic activation.

In Assay 1, following a 3-hour treatment with metabolic activation, cytotoxicity was seen at concentration range of 60 - 100 μg/mL. The 100 and 90 μg/mL concentrations showed marked cytotoxicity (relative total growth (RTG): 3 % and 9 %, respectively) and they were excluded from the evaluated concentration range. Thus, an evaluation was made using data of six concentrations (concentration range of 10 - 80 μg/mL). Relative total growth of the highest evaluated concentration (80 μg/mL) showed proper degree of cytotoxicity (RTG was 19 %).

There was statistically significant increase in the mutation frequency value of the four highest evaluated concentrations (40 - 80 μg/mL), the difference between the calculated values and the control exceed the Global Evaluation Factor, GEF (thus showing biological relevance) in the concentrations of 80, 70 and 60 μg/mL. At 40 μg/mL and lower concentrations the mutation frequency value was below the GEF. A concentration related increase was indicated by the linear trend analysis.

This experiment was considered as being a positive, meeting the criteria of positivity.

In Assay 1, following a 3-hour treatment without metabolic activation, cytotoxicity of the test material was observed at higher concentrations (concentration range of 70 - 100 μg/mL), lower degree of cytotoxicity was observed at 60 μg/mL (RTG value was 66 %) and no significant cytotoxicity was observed at lower concentrations. An evaluation was made using data of eight concentrations (concentration range of 10 - 100 μg/mL). Relative total growth of the highest evaluated and tested concentration (100 μg/mL) was 26 %. No statistically significant or biologically relevant increase in the mutation frequency was noted at any of the evaluated concentrations. Concentration related increase was indicated by the linear trend analysis. However, based on the individual values the difference between the calculated values and the control did not exceed the Global Evaluation Factor, GEF, thus it was not biologically relevant. This experiment was considered as being negative.

In Assay 2, following a 3-hour treatment with metabolic activation, cytotoxicity was seen at concentration range of 60 - 100 μg/mL, lower degree of cytotoxicity was observed at 40 μg/mL (RTG value was 61 %) and no significant cytotoxicity was observed at lower concentrations.

An evaluation was made using data of eight concentrations (concentration range of 10 - 100 μg/mL). Relative total growth of the highest evaluated and tested concentration (100 μg/mL) was 36 %. There was statistically significant increase in the mutation frequency value of the six highest evaluated concentrations (40 - 100 μg/mL), the difference between the calculated values and the control exceed the Global Evaluation Factor, GEF (thus showing biological relevance) in the concentrations of 100, 90, 80, 70 and 60 μg/mL. At 40 μg/mL or lower concentrations the mutation frequency value was below the GEF. A concentration related increase was indicated by the linear trend analysis. Lower degree of cytotoxicity was observed at the three highest concentrations in Assay 2 compared to Assay 1, however reproducibility was observed at the evaluated concentrations and the effect was reproducible between assays. Therefore, this experiment was considered to be reproducibly positive.

In Assay 2, following a 24-hour treatment without metabolic activation, cytotoxicity of the test material was observed at concentration range of 30 - 55 μg/mL. No cells survived the expression period in the samples of 55 and 50 μg/mL concentrations and excessive cytotoxicity was observed at 45 and 40 μg/mL concentrations. An evaluation was made using data of five concentrations (concentration range of 5 - 40 μg/mL). Relative total growth of the highest evaluated concentration (40 μg/mL was 1 %) and the RTG value of the next evaluated concentration (30 μg/mL) was 25 %, thus the range was covered between 1 % and 25 %. There was statistically significant increase in the mutation frequency values in the highest evaluated concentration in Assay 2 and concentration related increase was also indicated by the linear trend analysis. Based on the individual values the difference between the calculated values and the control exceed the Global Evaluation Factor, GEF, thus it was biologically relevant. However, this effect was seen only at the highest evaluated dose (which was used for evaluation for cytotoxicity data interpretation) at an excessive cytotoxicity level, therefore this result would not be considered positive. Furthermore, if this concentration (40 μg/mL) is excluded from the evaluation no statistically significant or biologically relevant increase in the mutation frequency was observed and no concentration related increase was indicated by the linear trend analysis. This experiment was considered as being negative.

The experiments were performed using appropriate untreated, negative (vehicle/solvent) and positive control samples in all cases. The spontaneous mutation frequency of the negative (vehicle/solvent) controls was in the appropriate range. The positive controls gave the anticipated increases in mutation frequency over the controls. The plating efficiencies for the negative (vehicle) controls at the end of the expression period were considered to be acceptable in all assays. The evaluated concentration ranges were considered to be adequate. The number of test concentrations met the acceptance criteria. Therefore, the study was considered to be valid.

Under the conditions of the study, treatment with the test material did result in a statistically significant and biologically relevant, dose dependent increase in the mutation frequency in the presence of a rat metabolic activation system (S9 fraction) in Assay 1, the observed effect was repeatable within or between assays. Therefore, reproducible positive effect of the test material was concluded in the performed experiments; overall the test material was considered to be positive with metabolic activation.

Negative results were seen in the experiment in the absence of a rat metabolic activation system (S9 fraction). Statistical differences were not supported by any results above the GEF with the exception of one concentration in Assay 2 without metabolic activation, however in this case excessive cytotoxicity was observed, thus the result would not be considered positive.

The Mouse Lymphoma Assay was considered to be valid and to reflect the real potential of the test material to cause mutations in the cultured mouse cells used in this study.

Overall the test material was considered to be mutagenic.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

It is proposed that a comet assay (in liver) combined with bone marrow micronucleus evaluation is an appropriate and scientifically justified assay for in vivo evaluation of the genotoxicity hazard identified for the substance in vitro.

Link to relevant study records

Reference

Endpoint:

genetic toxicity in vivo, other

Remarks:

in vivo mammalian somatic cell study: combined gene mutuation and cytogenicity / micronucleus assay

Type of information:

experimental study planned

Study period:

To be determined upon receipt of ECHA final decision

Justification for type of information:

TESTING PROPOSAL ON VERTEBRATE ANIMALS
Please see attached justification.

NON-CONFIDENTIAL NAME OF SUBSTANCE:
- Name of the substance on which testing is proposed to be carried out: Hydroxybenzaldehyde polymer with phenol (EC 947-320-5).

CONSIDERATIONS THAT THE GENERAL ADAPTATION POSSIBILITIES OF ANNEX XI OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION:
- Available GLP studies:
The following genotoxicity data have been generated to support an Annex VIII registration of the substance:
A bacterial reverse mutation test (OECD TG 471): Reported to be negative.
An in vitro CHO chromosome aberration test (OECD TG 473): Reported to be negative.
An in vitro mouse lymphoma assay (OECD TG 490): Reported to be positive in the presence of S9.
The REACh information requirements state that for substances demonstrated to be positive for any of the genotoxicity studies in Annex VII or VIII, appropriate in vivo mutagenicity assays shall be considered to ascertain if the genotoxicity potential observed in vitro can be expressed in vivo. Consequently, the attached testing proposal describes the applicant’s intended follow-up study, together with the justification for the choice of assay and its experimental design.
- Available non-GLP studies: There are no non-GLP studies available on the registered substance.
- Historical human data: There is no relevant historical human data available on the registered substance.
- (Q)SAR: QSAR is not appropriate for this UVCB substance.
- In vitro methods: It is not possible to conclude whether hazards identified in vitro will be expressed in vivo.
- Weight of evidence: A weight of evidence assessment of available in vitro data is presented in the attached testing proposal in order to determine the choice of the proposed in vivo study ans its experimental design.
- Grouping and read-across: No suitable surrogate substance has been identified for read-across.

CONSIDERATIONS THAT THE SPECIFIC ADAPTATION POSSIBILITIES OF ANNEXES VI TO X (AND COLUMN 2 THEREOF) OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION:
Column 2 of REACH Annex VIII states that "Appropriate in vivo mutagenicity studies shall be considered in case of a positive result in any of the genotoxicity studies in Annex VII or VIII".
Testing with the registered substance produced a positive result in the Mouse Lymphoma Assay in the presence of S9, with an equivocal response also apparent in the absence of S9.
In the chromosome aberration study, the registered substance produced aberration frequencies that exceeded the laboratory’s HCD, though these findings did not display a concentration-relationship and were not reproduced. They were therefore considered biologically irrelevant and the reported negative conclusion is supported.
In order to fully evaluate the genotoxic hazard of the registered substance, it is considered justified to conduct an in vivo study.
It is proposed that a comet assay in the liver, combined with assessment of micronuclei in the bone marrow, is an appropriate and scientifically justified assay for in vivo evaluation of the genotoxicity hazard identified in vitro for the registered substance. Please see the attached for the full justification.

FURTHER INFORMATION ON TESTING PROPOSAL IN ADDITION TO INFORMATION PROVIDED IN THE MATERIALS AND METHODS SECTION:
The combined assay would be conducted as follows:
- Species: Rat (This species is more commonly used for the comet assay and most laboratories have more HCD in rat than mouse).
- Strain: Common laboratory strain, e.g., Han Wistar or Sprague Dawley. The strain will be determined by availability of suitable historical control data at the selected test facility.
- Sex: To be determined in a preliminary dose setting phase. Assuming no substantial differences in maximum tolerated dose (MTD) are identified between the sexes, the main experiment will be conducted in males only.
- Exposure route: Oral gavage (Oral exposure is a potential route of human exposure).
- Duration of treatment: 3 consecutive doses. Necropsy and tissue processing 3 hours after the final (3rd) administration, equivalent to 24 hours after the 2nd administration.
- Dose levels: 0.25x MTD, 0.5x MTD, MTD (Actual doses to be determined based on the results of a preliminary dose setting phase conducted in the same species/strain of animals and using the same dosing regimen, route and dose volume).
- Controls: Concurrent negative (vehicle) and positive controls.
- Group size: N=5-6 for all groups depending on standard practice at test facility. For the positive control N=3 would be acceptable providing the laboratory can demonstrate suitable proficiency.
- Comet analysis: Liver (Measurement of % tail intensity (% DNA in tail) of 150 cells per tissue, per animal. Hedgehog cells, excluded from comet analysis but frequency to be recorded and reported).
- Micronucleus assessment: Bone marrow smears (Number of immature and mature erythrocytes in a total of 500 erythrocytes to be determined for toxicity assessment. 4000 immature erythrocytes to be assessed for the presence of micronuclei).
- Additional assessments:
Terminal blood sample taken and analysed for clinical chemistry parameters as an indicator of liver effects.
Sample of liver from each treated rat and vehicle control to be preserved in formalin for possible histopathology assessment in the event of positive comet findings.
Terminal blood sample to be processed to plasma and retained frozen as a contingency. In the event of negative comet and micronucleus results and assuming systemic exposure cannot be confirmed from existing data, this plasma sample could be analysed for the presence of Hydroxybenzaldehyde polymer and/or its metabolites.
Please refer to the attached for the full details of the proposed study.

Qualifier:

according to guideline

Guideline:

OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)

Version / remarks:

A comet assay in the liver, combined with assessment of micronuclei in the bone marrow.

Deviations:

not applicable

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Ames Test, Váliczkó (2016)

The test material was tested for potential mutagenic activity in accordance with the standardised guidelines OECD 471, EU Method B13/14 and OPPTS 870.5100, under GLP conditions using the Bacterial Reverse Mutation Assay. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimurium TA98, TA100, TA1535 and TA1537) and the tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coli WP2 uvrA) in the presence and absence of a post mitochondrial supernatant (S9 fraction) prepared from the livers of phenobarbital/β-naphthoflavone-induced rats.

The study included a Preliminary Compatibility Test, a Preliminary Range Finding Test (Informatory Toxicity Test), an Initial Mutation Test (Plate Incorporation Method) and a Confirmatory Mutation Test (Pre-Incubation Method).

Based on the results of the Compatibility Test, the test material was dissolved in DMSO. Concentrations of 5 000, 2 500, 1 000, 316, 100, 31.6 and 10 μg/plate were examined in the Range Finding Test. Based on the results of the Range Finding Test, the test material concentrations in the Initial Mutation Test and Confirmatory Mutation Test for Salmonella typhimurium strains were 1 581, 500, 158.1, 50, 15.81, 5, 1.581 and 0.5 μg/plate and for Escherichia coli WP2 uvrA strain were 5 000, 1 581, 500, 158.1, 50, 15.81, 5 and 1.581 μg/plate.

In the Initial Mutation Test and Confirmatory Mutation Test none of the observed revertant colony numbers were above the respective biological threshold value. There were no reproducible dose-related trends and no indication of any treatment effect.

Inhibitory, cytotoxic effects of the test material were observed in the Initial Mutation Test at 1 581 and 500 μg/plate concentrations in Salmonella typhimurium TA98, TA100, TA1537 strains with and without metabolic activation, in Salmonella typhimurium TA1535 strain without metabolic activation; at 1 581 μg/plate concentration in Salmonella typhimurium TA1535 strain with metabolic activation and at 5 000 and 1 581 μg/plate concentrations in Escherichia coli WP2 uvrA strain with and without metabolic activation.

Similar but stronger inhibitory, cytotoxic effects of the test material were observed in the Confirmatory Mutation Test in all Salmonella typhimurium bacterial strains at 1 581, 500 and 158.1 μg/plate concentrations without metabolic activation and at 1 581 and 500 μg/plate concentrations with metabolic activation; in Escherichia coli WP2 uvrA strain without metabolic activation at 5 000, 1 581 and 500 μg/plate concentrations and at 5 000 and 1 581 μg/plate concentrations with metabolic activation.

Slight precipitate was observed in the Confirmatory Mutation Test in all tested strains with metabolic activation at the concentrations of 5 000 and/or 1 581 μg/plate.

The mean values of revertant colonies of the solvent control plates were in good correlation with the historical control data, the reference mutagens showed the expected increase in the number of revertant colonies, the viability of the bacterial cells was checked by a plating experiment in each test. At least five analysable concentrations were presented in all strains of the main tests. The tests were considered to be valid.

The reported data of this mutagenicity assay show that under the experimental conditions applied the test material did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Under the conditions of this study, the test material had no mutagenic activity in the applied bacterium tester strains.

Chromosome Aberration, Orosz (2019)

The clastogenic potential of the test material was investigated in vitro, in a study which was conducted in accordance with the standardised guideline OECD 473, under GLP condictions, using Chinese hamster V79 lung cells. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

During the study the test material was formulated in DMSO and it was examined up to cytotoxic concentrations. In independent Chromosome Aberration Assays using duplicate cultures, at least 300 well-spread metaphase cells (or until a clear positive response was detected) were analysed for each evaluated test material treated, negative (vehicle) and positive control sample.

In Chromosome Aberration Assay 1, a 3-hour treatment with metabolic activation (in the presence of S9-mix) and a 3-hour treatment without metabolic activation (in the absence of S9-mix) were performed. Sampling was performed 20 hours after the beginning of the treatment in both cases. The examined concentrations of the test material were 50, 40, 30, 25, 15 and 7.5 μg/mL (experiment with and without metabolic activation).

In Assay 1, insolubility was not detected with and without metabolic activation. There were no large changes in the pH and osmolality. Marked cytotoxicity was observed in the experiment with metabolic activation (RICC value of the highest evaluated concentration (40 μg/mL) with metabolic activation was 42 %). The same effect was observed in the experiment without metabolic activation (RICC value of the highest evaluated concentration (30 μg/mL) without metabolic activation was 41 %). Therefore, concentrations of 40, 15 and 7.5 μg/mL (a total of three) were chosen for evaluation in the experiment with metabolic activation, concentrations of 30, 15 and 7.5 μg/mL (a total of three) were chosen for evaluation in the experiment without metabolic activation.

In Chromosome Aberration Assay 2, a 3-hour treatment with metabolic activation (in the presence of S9-mix) and a 20-hour treatment without metabolic activation (in the absence of S9-mix) were performed. Sampling was performed 20 hours after the beginning of the treatment in both cases. The examined concentrations of the test material were 50, 40, 30, 25, 15 and 7.5 μg/mL (experiment with metabolic activation) and 25, 20, 15, 10, 5 and 2.5 μg/mL (experiment without metabolic activation).

In Assay 2 insolubility was not detected with and without metabolic activation. There were no large changes in the pH and osmolality. Marked cytotoxicity was observed in the experiment with metabolic activation (RICC value of the highest evaluated concentration (40 μg/mL) with metabolic activation was 45 %). The same effect was observed in the experiment without metabolic activation (RICC value of the highest evaluated concentration (20 μg/mL) without metabolic activation was 36 %). Therefore, concentrations of 40, 15 and 7.5 μg/mL (a total of three) were evaluated in the experiment with metabolic activation, and concentrations of 20, 10 and 5 μg/mL (a total of three) were evaluated in the experiment without metabolic activation.

None of the treatment concentrations caused a biologically or statistically significant increase in the number of cells with structural chromosome aberrations in either assay with or without metabolic activation when compared to the appropriate negative (vehicle) control values.

Polyploid metaphases and/or endoreduplicated metaphases were found in some cases in the negative (vehicle) control or positive control or test material treated samples in the performed experiments, but their incidence was not related to treatment with the test material.

The negative (vehicle) control data were within the acceptable range for the spontaneous aberration frequency, the positive control substances caused a statistically significant increase in the number of structural aberrations excluding gaps in the experiments with or without metabolic activation demonstrating the sensitivity of the test system. The evaluated concentration range was considered to be adequate; three test material treated concentrations were evaluated in each assay. The tests were considered to be valid.

In conclusion, under the conditions of the study the test material did not induce a significant level of chromosome aberrations in Chinese hamster V79 cells either with or without metabolic activation. Therefore, the test material was considered as not clastogenic in this test system.

Mouse Lymphoma Assay, Varga-Kanizsai (2020)

An in vitro mammalian cell assay was performed in mouse lymphoma L5178Y TK+/- 3.7.2 C cells at the tk locus to test the potential of the test material to cause gene mutation and/or chromosome damage. The study was conducted in accordance with the standardised guidelines OECD 490 and EU Method B.17, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

Treatment was performed for 3 hours with and without metabolic activation (±S9 mix) and for 24 hours without metabolic activation (-S9 mix).

Dimethyl sulfoxide (DMSO) was used as vehicle of the test material in this study. Based on the preliminary toxicity test, the following test material concentrations were examined in the mutation assays:

Assay 1, 3-hour treatment with and without metabolic activation: 100, 90, 80, 70, 60, 40, 20 and 10 μg/mL,

Assay 2, 3-hour treatment with metabolic activation: 100, 90, 80, 70, 60, 40, 20 and 10 μg/mL,

Assay 2, 24-hour treatment without metabolic activation: 55, 50, 45, 40, 30, 20, 10 and 5 μg/mL.

In Assays 1 - 2 with and without metabolic activation, there were no large changes in pH or osmolality after treatment. No insolubility was observed in the final treatment medium at the end of the treatment in Assays 1 - 2 with and without metabolic activation.

In Assay 1, following a 3-hour treatment with metabolic activation, cytotoxicity was seen at concentration range of 60 - 100 μg/mL. The 100 and 90 μg/mL concentrations showed marked cytotoxicity (relative total growth (RTG): 3 % and 9 %, respectively) and they were excluded from the evaluated concentration range. Thus, an evaluation was made using data of six concentrations (concentration range of 10 - 80 μg/mL). Relative total growth of the highest evaluated concentration (80 μg/mL) showed proper degree of cytotoxicity (RTG was 19 %).

There was statistically significant increase in the mutation frequency value of the four highest evaluated concentrations (40 - 80 μg/mL), the difference between the calculated values and the control exceed the Global Evaluation Factor, GEF (thus showing biological relevance) in the concentrations of 80, 70 and 60 μg/mL. At 40 μg/mL and lower concentrations the mutation frequency value was below the GEF. A concentration related increase was indicated by the linear trend analysis.

This experiment was considered as being a positive, meeting the criteria of positivity.

In Assay 1, following a 3-hour treatment without metabolic activation, cytotoxicity of the test material was observed at higher concentrations (concentration range of 70 - 100 μg/mL), lower degree of cytotoxicity was observed at 60 μg/mL (RTG value was 66 %) and no significant cytotoxicity was observed at lower concentrations. An evaluation was made using data of eight concentrations (concentration range of 10 - 100 μg/mL). Relative total growth of the highest evaluated and tested concentration (100 μg/mL) was 26 %. No statistically significant or biologically relevant increase in the mutation frequency was noted at any of the evaluated concentrations. Concentration related increase was indicated by the linear trend analysis. However, based on the individual values the difference between the calculated values and the control did not exceed the Global Evaluation Factor, GEF, thus it was not biologically relevant. This experiment was considered as being negative.

In Assay 2, following a 3-hour treatment with metabolic activation, cytotoxicity was seen at concentration range of 60 - 100 μg/mL, lower degree of cytotoxicity was observed at 40 μg/mL (RTG value was 61 %) and no significant cytotoxicity was observed at lower concentrations.

An evaluation was made using data of eight concentrations (concentration range of 10 - 100 μg/mL). Relative total growth of the highest evaluated and tested concentration (100 μg/mL) was 36 %. There was statistically significant increase in the mutation frequency value of the six highest evaluated concentrations (40 - 100 μg/mL), the difference between the calculated values and the control exceed the Global Evaluation Factor, GEF (thus showing biological relevance) in the concentrations of 100, 90, 80, 70 and 60 μg/mL. At 40 μg/mL or lower concentrations the mutation frequency value was below the GEF. A concentration related increase was indicated by the linear trend analysis. Lower degree of cytotoxicity was observed at the three highest concentrations in Assay 2 compared to Assay 1, however reproducibility was observed at the evaluated concentrations and the effect was reproducible between assays. Therefore, this experiment was considered to be reproducibly positive.

In Assay 2, following a 24-hour treatment without metabolic activation, cytotoxicity of the test material was observed at concentration range of 30 - 55 μg/mL. No cells survived the expression period in the samples of 55 and 50 μg/mL concentrations and excessive cytotoxicity was observed at 45 and 40 μg/mL concentrations. An evaluation was made using data of five concentrations (concentration range of 5 - 40 μg/mL). Relative total growth of the highest evaluated concentration (40 μg/mL was 1 %) and the RTG value of the next evaluated concentration (30 μg/mL) was 25 %, thus the range was covered between 1 % and 25 %. There was statistically significant increase in the mutation frequency values in the highest evaluated concentration in Assay 2 and concentration related increase was also indicated by the linear trend analysis. Based on the individual values the difference between the calculated values and the control exceed the Global Evaluation Factor, GEF, thus it was biologically relevant. However, this effect was seen only at the highest evaluated dose (which was used for evaluation for cytotoxicity data interpretation) at an excessive cytotoxicity level, therefore this result would not be considered positive. Furthermore, if this concentration (40 μg/mL) is excluded from the evaluation no statistically significant or biologically relevant increase in the mutation frequency was observed and no concentration related increase was indicated by the linear trend analysis. This experiment was considered as being negative.

The experiments were performed using appropriate untreated, negative (vehicle/solvent) and positive control samples in all cases. The spontaneous mutation frequency of the negative (vehicle/solvent) controls was in the appropriate range. The positive controls gave the anticipated increases in mutation frequency over the controls. The plating efficiencies for the negative (vehicle) controls at the end of the expression period were considered to be acceptable in all assays. The evaluated concentration ranges were considered to be adequate. The number of test concentrations met the acceptance criteria. Therefore, the study was considered to be valid.

Under the conditions of the study, treatment with the test material did result in a statistically significant and biologically relevant, dose dependent increase in the mutation frequency in the presence of a rat metabolic activation system (S9 fraction) in Assay 1, the observed effect was repeatable within or between assays. Therefore, reproducible positive effect of the test material was concluded in the performed experiments; overall the test material was considered to be positive with metabolic activation.

Negative results were seen in the experiment in the absence of a rat metabolic activation system (S9 fraction). Statistical differences were not supported by any results above the GEF with the exception of one concentration in Assay 2 without metabolic activation, however in this case excessive cytotoxicity was observed, thus the result would not be considered positive.

The Mouse Lymphoma Assay was considered to be valid and to reflect the real potential of the test material to cause mutations in the cultured mouse cells used in this study.

Overall the test material was considered to be mutagenic.

In vivo

There is currently no available in vivo genotoxicity information for the substance.

The registrant is proposing to carry out an in vivo mutagenicity study. It is proposed that a comet assay (in liver) combined with bone marrow micronucleus evaluation is an appropriate and scientifically justified assay for in vivo evaluation of the genotoxicity hazard identified for the substance in vitro.

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008, the substance does not require classification with respect to genetic toxicity.